This proposal is Project 2 within a P01 renewal, entitled Intracellular pathogens and innate immunity. M. tuberculosis is an important human pathogen that causes severe morbidity and mortality around the world. The goal of the previous proposal was to identify the mechanisms by which M. tuberculosis triggers and manipulates host responses of its primary host cell, the macrophage. We made the striking discovery that M. tuberculosis, a phagosomal pathogen, activates the host cytosolic surveillance pathway (CSP), an innate signaling pathway that senses bacterial molecules in the cytoplasm. We are interested in host and bacterial factors required for CSP activation, and in elucidating the functional role of the pathway in M. tuberculosis pathogenesis. We have shown that the ESX-1 secretion pathway and the cell wall lipid PDIM are critical for perturbing phagosomal membranes of macrophages, allowing activation of cytosolic DNA receptors. Unexpectedly, cytosolic access also targets bacteria to the autophagy pathway. We hypothesize that intracellular pathogens perturb intracellular membranes to promote virulence, but activation of the CSP allows host cells to discriminate and mount qualitatively different immune responses to pathogens versus non-pathogens. In Aim 1, we propose to elucidate the mechanism by which M. tuberculosis gains access to the cytosol and activates the CSP, building upon our evidence that a single ESX-1 substrate, ESAT-6, functions to permeabilize the phagosomal membrane. We propose to identify the nature of the DNA that is recognized by the host, test the role of putative host receptors responsible for CSP activation, and probe the role of cytosolic signaling in M. tuberculosis infection. In Aim 2, we will examine how cytosolic access leads to targeting of M. tuberculosis to the autophagy pathway. In particular, we will test the role of ubiquitination and ubiquitin-binding adapters in targeting of autophagic vesicles to M. tuberculosis during infection. In the final Aim, We will collaborate extensively with the Portnoy (Project 1) and Vance (Project 3) groups to screen for modulators of M. tuberculosis growth and host innate responses by carrying out a screen in macrophages isolated from ENU mutagenized mice, and by performing an RNAi screen in macrophages.